Method of separating protective components of bordetella pertussis

ABSTRACT

To provide a method of efficiently separate protective components of  Bordetella pertussis . On the basis of differences in adsorbability to calcium phosphate gel formed by adding calcium ions to a  Bordetella pertussis  culture in the presence of excess phosphate ions, protective components of  Bordetella pertussis  are separated from the  Bordetella pertussis  culture. Traditionally, protective components of  Bordetella pertussis  have been separated using different purification methods for the respective components. According to the present invention, the use of the same means of purification for all subject components makes it possible to purify each component with high efficiency and high recovery rate, an aspect very advantageous for industrial production. It is also possible to efficiently produce an improved purified pertussis component vaccine comprising an effective combination of pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT).

This application is a continuation of application Ser. No. 11/105,540,filed Apr. 14, 2005, which is a continuation of application Ser. No.10/206,989 filed Jul. 30, 2002, now abandoned, which is a continuationof application Ser. No. 09/349,712 filed Jul. 8, 1999, now abandoned,which is a continuation of application Ser. No. 08/530,373, filed Oct.13, 1995, now U.S. Pat. No. 6,051,240, which is a National PhaseApplication of application No. PCT/JP95/00830, filed Apr. 26, 1995,published in a non-English language. The entire contents of each of theaforementioned applications are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method of separating protectivecomponents of Bordetella pertussis. The pertussis component vaccine canbe produced by suitably mixing the protective components separated bythe method of the present invention.

BACKGROUND ART

Vaccines are widely used to prevent communicable diseases. Pertussis, acommunicable respiratory disease caused by infection with Bordetellapertussis, is likely to severely affect patients, especially infants,due to apneic cough with occasional spasm. To cope with this disease, ithas been common practice to use whole cultured cells of Bordetellapertussis after inactivation (inactivated vaccine). However, localizedreactions at the site of vaccination and side reactions, such as fever,have been reported, creating a social urge to solve this problem. Tosolve this problem, there have been a large number of attempts of usingprotective components separated from Bordetella pertussis as vaccine.For example, acellular pertussis vaccine (ACP vaccine), prepared byextracting protective proteins, such as pertussis toxin (PT), pertussisfilamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussisfimbriae (FIM), from Bordetella pertussis cells, and removing endotoxin(ET), is being into practical application, but is not fullysatisfactory, due to the drawbacks described below.

Pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA),pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM), all protectivecomponents of Bordetella pertussis already in practical application withvalidated efficacy, are separated by respective methods.

Pertussis toxin (PT) can be separated by affinity chromatography usinghuman haptoglobin as a ligand [Biochimica et Biophysica Acta, Vol. 580,p. 175 (1979)]. However, human haptoglobin can be contaminated withhepatitis virus, because it is collected from human blood; the sameapplies when animal sera are used. Another available method is affinitychromatography using denatured ceruloplasmin as a ligand (JapanesePatent Unexamined Publication No. 62135/1987). Although this method isfree of the problem of viral contamination, some problems arise,including vaccine contamination with ceruloplasmin and the high toxicityand potential body retention of sodium thiocyanate and other eluentshaving protein-denaturing effect.

As for pertussis filamentous hemagglutinin (FHA), a purification methodusing hydroxyapatite gel is available [Infection and Immunity, Vol. 41,p. 313 (1983) and EP-A-231083, EP-A-427462, EP-A-462534; Japanese PatentUnexamined Publication Nos. 234031/1987, 169893/1992, 368337/1993).However, it takes long time for column operation, and is uneconomic dueto the high cost of hydroxyapatite.

As for pertactin (PRN, 69K-OMP), affinity chromatography using a mouseserum as a ligand is available [Infection and Immunity, Vol. 56, p. 3189(1988)], but has the same drawbacks as above.

As for pertussis fimbriae (FIM), Bordetella pertussis cell extract ispurified by salting-out with ammonium sulfate and magnesium chloride[Infection and Immunity, Vol. 48, p. 442 (1985)], but this method ispoor in vaccine production efficiency due to low yield.

There is a method of preparing Gram-negative bacterial vaccine byadosorbing with the aluminum hydroxide gel (WO 93/10216). This methodneeds the large amount of the aluminum hydroxide gel, which adsorbs boththe protective components and the endotoxin originated in Gram-negativebacteria. The vaccine obtained by the method of WO93/10216 has a dangerof side effects, such as fever and endotoxin-shock, by the endotoxinreleased into body because of the diluted vaccines.

As for the pertussis vaccine production included as the componentsmixture without separating each protective component originated inBordetella pertussis, a method of using calcium phosphate gel isavailable (EP-A-291968, Japanese Patent Unexamined Publication No.52726/1989). However, this method formed the calcium phosphate in thepresence of a 1M sodium chloride does not absorb the protectivecomponents.

As stated above, totally different purification methods must be used toseparate the respective protective components of Bordetella pertussis.This approach is unsuitable to large-scale vaccine production due topainstaking operation, and difficult to apply practically. Moreover, thecustomary methods of separating protective components disclosed in priorart have some problems that materials or reagents have pathogenicity ortoxity.

DISCLOSURE OF INVENTION

Against the background described above, the present inventorsinvestigated methods of efficiently separating protective components ofBordetella pertussis, and found that protective components of Bordetellapertussis can be efficiently separated from Bordetella pertussis cultureon the basis of differences in adsorbability to calcium phosphate gelformed by adding calcium ions to the Bordetella pertussis culture in thepresence of excess phosphate ions. The inventors made furtherinvestigation based on this finding, and the efficient and safety methodof separating the protective components combined with the calciumphosphate gel treatment and elution by salt and heating was developedthe present invention. Accordingly, the present invention relates to:

(1) A method of separating at least one member of the group consistingof pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP),pertussis fimbriae (FIM), and pertussis toxin (PT) by bringing aBordetella pertussis culture into contact with calcium phosphate gelwhich is formed by adding calcium ions to the culture in the presence ofphosphate ions.

(2) A method of separating at least one member of the group consistingof pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP),pertussis fimbriae (FIM) and pertussis toxin (PT) by separating aBordetella pertussis culture into cells and culture liquid, and carryingout at least one of processes (A), (B), (C) and (D):

(A) a process in which the separated cells are eluted with a saltsolution, and pertussis filamentous hemagglutinin (FHA) is separated bybringing the eluted solution into contact with calcium phosphate gel ofthe above item (1),(B) a process in which the cell residue resulting from the elutiontreatment of the above process (A) is heated in the presence of a saltsolution and brought into contact with calcium phosphate gel, andpertactin (PRN, 69K-OMP) is separated by bringing the eluted solutioninto contact with calcium phosphate gel of the above item (1),(C) a process in which the cell residue resulting from the elutiontreatment of the above process (A) is heated in the presence of a saltsolution, the supernatant is brought into contact with calcium phosphategel and eluted with a salt solution, and pertussis fimbriae (FIM) isseparated by bringing the eluted solution into contact with calciumphosphate gel of the above item (1),(D) a process in which the culture or the separated culture liquid isbrought into contact with calcium phosphate gel of the above item (1),and pertussis toxin (PT) is separated from the supernatant.

(3) The separation method of the above item (2), wherein the supernatantis brought into contact with calcium phosphate gel and eluted with asalt solution to separate pertussis filamentous hemagglutinin (FHA) inprocess (A).

(4) The separation method of the above item (2), wherein the supernatantafter being brought into contact with calcium phosphate gel is broughtinto contact with ion exchange gel to separate pertactin (PRN, 69K-OMP)in process (B).

(5) The separation method of the above item (2), wherein the supernatantis brought into contact with calcium phosphate gel and removed, and theresulting residue is eluted with a salt solution to separate pertussisfimbriae (FIM) in process (C).

(6) The separation method of the above item (2), wherein the supernatantis brought into contact with ion exchange gel to separate pertussistoxin (PT) in process (D).

(7) The separation method of the above item (2), wherein the saltsolution used in processes (A) and (C) is a buffer containing an alkalimetal salt.

(8) The separation method of the above item (7), wherein the saltsolution is a buffer containing 0.01-1.0 M sodium chloride.

(9) The separation method of the above item (1) or (2), wherein thecalcium phosphate gel is formed by adding calcium ions to the culture orthe supernatant of pH 7-9 in the presence of phosphate ions.

(10) The separation method of the above item (9), wherein the equivalentratio of phosphate ions and calcium ions is 1.25-30 equivalents ofphosphate ions per equivalent of calcium ions.

(11) The separation method of the above item (9), wherein the calciumphosphate gel is formed by adding calcium acetate, as a calcium ionsource, at 0.1-2 w/v % in the presence of a 0.05-0.1 M phosphate buffer.

(12) The separation method of the above item (1) or (2), wherein atleast one member of the group consisting of pertussis toxin (PT),pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) andpertussis fimbriae (FIM) is separated, after which endotoxin is removedby adsorption to aluminum hydroxide gel in the presence of ammoniumsulfate.

(13) The separation method of the above item (1) or (2), wherein atleast one member of the group consisting of pertussis toxin (PT),pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) andpertussis fimbriae (FIM) is separated, after which endotoxin is removedby zonal centrifugation.

(14) A pertussis vaccine wherein the components PT:FHA:FIM are admixedin a ratio of 4-6:8-10:1.

(15) A pertussis vaccine wherein the components PT:FHA:PRN:FIM areadmixed in a ratio of 2-6:4-10:1-2:1.

BEST MODE FOR CARRYING OUT THE INVENTION

The Bordetella pertussis strain used for the present invention is notsubject to limitation, as long as it is capable of producing one or morethan one member of the group consisting of pertussis filamentoushemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM)and pertussis toxin (PT), all protective components of Bordetellapertussis. Useful strains include known strains, such as Bordetellapertussis Tohama phase I strain [Infection and Immunity, Vol. 6, p. 89,(1972)] (maintained at the National Institute of Health, Ministry ofSocial Welfare, Tokyo, Japan (NIHJ 1052), deposited under accessionnumber IFO 14073 at the Institute for Fermentation, Osaka since Aug. 13,1980), Bordetella pertussis Yamaguchi phase I strain, Bordetellapertussis phase I strain 18-323 and Bordetella pertussis phase I strain165, with preference given to Bordetella pertussis Tohama phase I strain(IFO 14073) from the viewpoint of productivity. Bordetella pertussis canbe cultured by known methods. Useful media include known basal media,such as Cohen-Wheeler medium, Stainer-Scholte medium and other liquidmedia, with preference given to Stainer-Scholte medium. The solutioncontaining protective components and endotoxin (ET) may be a cultureobtained by stationary culture or tank culture. In the presentinvention, the culture means cultured cells or culture liquid resultingfrom incubating said Bordetella pertussis. And the present invention,the supernatant means the culture liquid or the supernatant resultingfrom heating the cells in the presence of a salt solution or elutingwith a salt solution from the calcium phosphate gel adsorbed theprotective components as described below. The cells include the culturecells and the cell residue. In the present invention, the method used toseparate a Bordetella pertussis culture into cells and culturesupernatant may be a known method, such as centrifugation or filtration.

The calcium phosphate gel used for the present invention is not aready-made gel, but preferably calcium phosphate gel formed in a cultureor a supernatant to be treated by adding calcium ions to them in thepresence of excess phosphate ions (may referred to as the in-side gelforming method). Although prepared calcium phosphate gel (e.g.,commercially available hydroxyapatite gel). In comparison with theformer method by using the ready-made hydroxyapatite gel mentioned above(may referred to as the out-side gel forming method), the present methodby using calcium phosphate gel is higher in both adsorption efficiencyfor pertussis filamentous hemagglutinin (FHA) and pertussis fimbriae(FIM) and recovery efficiency of them, as shown hereafter. Moreover, thecalcium phosphate gel used in the present invention is better inoperational efficiency because of the absence of gel pretreatment andregeneration process, and more advantageous in cost. Furthermore, eachof protective components of Bordetella pertussis can be selectivelyabsorbed to the calcium phosphate gel by properly selecting the ratio ofphosphate ions to calcium ions. If the culture or the supernatant to betreated with calcium phosphate gel, does not contain a sufficient amountof phosphate ions, a phosphate buffer of appropriate concentration isadded to provide phosphate ions before addition of calcium ions. Forexample, by adding 1 M phosphate buffer, the final phosphate ionconcentration is adjusted to 0.02-0.2 M, preferably 0.05-0.1 M.

The calcium ion source added is exemplified by soluble calcium salts,such as calcium acetate, calcium chloride and calcium nitrate, withpreference given to calcium ions derived from calcium acetate.Concerning the ratio of phosphate ions and calcium ions, it ispreferable that phosphate ions be in excess, in comparison with calciumions. The ratio can be properly selected in each case of the protectivecomponents of Bordetella pertussis, as mentioned hereafter.

In process (A) above, pertussis filamentous hemagglutinin (FHA) isseparated as follows: After the culture liquid, i.e. the culturesupernatant, is removed from a Bordetella pertussis culture by a knownmethod, such as centrifugation or filtration, a one-tenth toone-twentieth volume (relative to the amount of culture broth)(corresponding to a final cell concentration of 50-100 billion cells/ml)of a salt solution is added to the cells to elute the hemagglutinin. Inthis case, the salt solution used is preferably a buffer supplementedwith an alkali metal salt or an alkaline earth metal salt, specificallya 0.04-0.08 M phosphate buffer supplemented with a 0.25-1.0 M alkalimetal salt or alkaline earth metal salt, with greater preference givento a 0.05 M phosphate buffer supplemented with a 0.5-1.0 M alkali metalsalt. The alkali metal salt or alkaline earth metal salt added to thebuffer is exemplified by sodium chloride, potassium chloride andmagnesium chloride. For example, it is preferable to elute thehemagglutinin by adding a one-tenth to one-twentieth volume (relative tothe amount of culture broth) of a 0.04-0.08 M phosphate buffer (pH 7-9)supplemented with 0.5-1.0 M sodium chloride, more preferably a 0.05 Mphosphate buffer (pH 8) supplemented with 1 M sodium chloride, to thecells collected, followed by gentle stirring at 4° C. to roomtemperature, preferably 8-15° C., for 1-60 minutes, preferably 1-30minutes, and standing for 1-2 days. The solution containing the elutedpertussis filamentous hemagglutinin (FHA) is then subjected to a knownmethod, such as centrifugation or filtration, to recover the supernatant(the cell residue obtained at the same time by this treatment is used toisolate pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM)). Thethus-obtained supernatant is then brought into contact with calciumphosphate gel.

Concerning the ratio of phosphate ions and calcium ions, it ispreferable that phosphate ions be in excess, in comparison with calciumions. For example, the equivalent ratio of these ions is preferably1.25-30 equivalents, more preferably 1.5-7.5 equivalents of phosphateions per equivalent of calcium ions. This quantitative ratio can beexpressed in molar ratio as 0.8-20 M of phosphate ions to 1 M of calciumions, more preferably 1-5 M of phosphate ions to 1 M of calcium ions.For example, to a solution (pH 7-9) containing phosphate ions at aconcentration within the above-described concentration range (0.02-0.2M, preferably 0.05-0.1 M), a calcium salt is added to a finalconcentration of 4-70 mM, preferably 8-50 mM (e.g., calcium acetateadded to a final concentration of 0.1-0.8 w/v %, preferably 0.2-0.6 w/v%), followed by gentle reaction at 4° C. to room temperature, preferably8-15° C., for 1 to 4 hours, preferably 1 to 2 hours, to form calciumphosphate gel.

Although pertussis filamentous hemagglutinin (FHA) is adsorbed to thecalcium acetate gel added, provided that the amount of calcium phosphategel added to a final concentration exceeding 0.8 w/v %, it is preferableto add the calcium acetate gel in an amount such that the finalconcentration falls within the above concentration range, forselectively adsorbing pertussis filamentous hemagglutinin (FHA) only.After completion of the reaction, the supernatant is removed by a knownmethod, such as centrifugation or filtration; the resulting gelprecipitate is collected. To this precipitate, a one-tenth toone-twentieth volume (relative to the amount of culture broth) of a saltsolution is added, to elute the pertussis filamentous hemagglutinin(FHA). In this case, the salt solution used may be the same saltsolution as used to elute pertussis filamentous hemagglutinin (FHA) fromthe above-described cells. It is preferable to add a one-tenth toone-twentieth volume of 0.05-0.1 M phosphate buffer (pH 7-9)supplemented with 1-2 M sodium chloride, more preferably 0.1 M phosphatebuffer (pH 8) supplemented with 1-1.5 M sodium chloride, to theabove-described gel precipitate, followed by gentle stirring at 4° C. toroom temperature for 1 to 2 hours, to elute the hemagglutinin. Aftercompletion of the stirring, the precipitate is removed by a knownmethod, such as centrifugation or filtration, to recover pertussisfilamentous hemagglutinin (FHA) in the supernatant. The supernatant, ifnecessary, can be concentrated and desalinized, by ammonium sulfatesalting-out or using an ultrafiltration membrane. By subjecting thesupernatant obtained by the above-described treatment to the aluminumhydroxide gel treatment or zonal centrifugation described below,pertussis filamentous hemagglutinin (FHA) having endotoxin selectivelyremoved can be separated with substantially no loss.

In process (B) or (C) above, pertactin (PRN, 69K-OMP) and pertussisfimbriae (FIM) are separated as follows: The cell residue resulting fromelution of the solution containing pertussis filamentous hemagglutinin(FHA) is heated in the presence of a one-tenth to one-twentieth volume(relative to the amount of culture broth) (corresponding to a final cellconcentration of 500-100 billion cells/ml) of a salt solution to extractthe pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM). In this case,the salt solution used may be the same as used in process (A) above.However, it is preferable to use a one-tenth to one-twentieth volume(relative to the amount of culture broth) of 0.01-0.05 M phosphatebuffer (pH 7-9) supplemented with 0.15-0.25 M sodium chloride, withgreater preference given to 0.01 M phosphate buffer (pH 7) supplementedwith 0.15-0.25 M sodium chloride. It is preferable that heating beachieved in warm water at 40-80° C., preferably 50-60° C., for 60 to 120minutes, preferably 80 to 90 minutes. The heated extracted pertactin(PRN, 69K-OMP) and pertussis fimbriae (FIM) are recovered in thesupernatant by a known method, such as centrifugation or filtration. Thethus-obtained supernatant is then brought into contact with calciumphosphate gel. In this case, calcium phosphate gel treatment can beperformed in accordance with process (A) above; however, it ispreferable to perform it within the following concentration range. Forexample, to a solution (pH 7-9) containing phosphate ions, adjusted asnecessary to a final phosphate ion concentration of 0.05-0.1 M,preferably 0.1 M, by adding a 1 M phosphate buffer, or the like, acalcium salt is added to a final concentration of 40-180 mM, preferably55-150 mM (e.g., calcium acetate added to a final concentration of 1-2w/v %, preferably 1.3-1.7 w/v %), followed by gentle reaction at 4° C.to room temperature, preferably 8-15° C., for 1 to 4 hours, preferably 1to 2 hours, to form calcium phosphate gel. After completion of thereaction, the resulting precipitate and supernatant are separated fromeach other by a known separation method, such as filtration orcentrifugation, to recover pertactin (PRN, 69K-OMP) in the supernatantand pertussis fimbriae (FIM) in the gel residue, with substantially noloss.

The crudely purified pertactin (PRN, 69K-OMP) obtained by theabove-described treatment can be further purified by a known method,preferably by ion exchange gel treatment; it is preferable that thecrudely purified pertactin (PRN, 69K-OMP) be previously concentrated anddesalinized by ammonium sulfate salting-out or using an ultrafiltrationmembrane. In the present invention, useful ion exchange gels includeanion exchange gel and cation exchange gel, with preference given tocation exchange gel. Contact with ion exchange gel may be achieved bythe column chromatography method or the batch method. By this treatment,impurities, i.e., substances other than pertactin (PRN, 69K-OMP) in thecrudely purified pertactin (PRN, 69K-OMP), are adsorbed; the effluent iscollected to yield a solution containing pertactin (PRN, 69K-OMP). Inthe column chromatography method, the column is packed with ion exchangegel, through which the starting material, i.e., crudely purifiedpertactin (PRN, 69K-OMP), is passed at a flow rate of 100-500 ml/cm²/hr.In the batch method, crudely purified pertactin (PRN, 69K-OMP) is placedin a container, to which ion exchange gel is added directly, followed bystirring for about 30 minutes to 3 hours, preferably about 1 hour, toadsorb impurities, i.e., substances other than pertactin (PRN, 69K-OMP).Such impurity adsorption is achieved using a buffer of a pH value of5.0-8.0 and an electroconductivity of 100-300 umho (0.1-0.3 mS), e.g., a0.01-0.02 M phosphate buffer (pH 5.5-6.0). By subjecting the supernatantobtained by the above-described treatment to the aluminum hydroxide geltreatment or zonal centrifugation treatment described below, pertactin(PRN, 69K-OMP) having endotoxin removed can be separated withsubstantially no loss.

To the gel residue containing crude pertussis fimbriae (FIM) obtained bythe above-described treatment, a one-tenth to one-twentieth volume(relative to the amount of culture broth) of a salt solution is added,to elute the pertussis fimbriae (FIM). In this case as well, the saltsolution may be the same as used in process (A) above. For example, itis preferable to add a one-tenth to one-twentieth volume (relative tothe amount of culture broth) of a 0.05-0.1 M phosphate buffer (pH 7-9)supplemented with 1-2 M sodium chloride, preferably a 0.1 M phosphatebuffer (pH 8) supplemented with 1-1.5 M sodium chloride, followed bygentle stirring at 4° C. to room temperature for 1 to 2 hours, to elutethe pertussis fimbriae (FIM). After completion of the stirring, theprecipitate is removed by a known method, such as centrifugation orfiltration, to recover pertussis fimbriae (FIM) in the supernatant. Bysubjecting the supernatant obtained by the above-described treatment tothe above-described aluminum hydroxide gel treatment or zonalcentrifugation treatment, pertussis fimbriae (FIM) having endotoxinselectively removed can be separated with substantially no loss.

In process (D) above, pertussis toxin (PT) is separated as follows:Although a Bordetella pertussis culture can be used without separationinto cultured cells and culture supernatant in this process, it ispreferable in respect of efficiency to recover the supernatant from theBordetella pertussis culture by a known method, such as centrifugationor filtration, concentrate the supernatant about 10-20 fold using anultrafiltration membrane, or the like, and collect the supernatant bycentrifugation or another method before this process. This supernatantis then brought into contact with calcium phosphate gel. In this case,calcium phosphate gel treatment can be carried out in the same manner asin process (A) above, but it is preferable to carried out this treatmentwithin the following concentration range. For example, to a solution (pH7-9) containing phosphate ions, adjusted as necessary to a finalphosphate ion concentration of 0.05-0.1 M, preferably 0.1 M, by adding a1 M phosphate buffer, or the like, a calcium salt is added to a finalconcentration of 40-180 mM, preferably 55-150 mM (e.g., calcium acetateadded to a final concentration of 1-2 w/v %, preferably 1.3-1.7 w/v %),followed by gentle reaction at 4° C. to room temperature, preferably8-15° C., for 1 to 4 hours, preferably 1 to 2 hours, to form calciumphosphate gel. After completion of the reaction, the resultingprecipitate and supernatant are separated from each other by a knownmethod, such as centrifugation or filtration, to recover pertussis toxin(PT) in the supernatant with substantially no loss. The crudely purifiedpertussis toxin (PT) obtained by the above-described treatment isfurther purified by ion exchange gel treatment; it is preferable thatthe crudely purified pertussis toxin (PT) be previously concentrated anddesalinized by ammonium sulfate salting-out or using an ultrafiltrationmembrane. The ion exchange gel used here is exemplified by anionexchange gel and cation exchange gel, with preference given to cationexchange gel. Contact with ion exchange gel may be achieved by thecolumn chromatography method or the batch method. By this treatment,pertussis toxin (PT) in the crudely purified pertussis toxin (PT) isadsorbed to the gel, followed by washing with an appropriate buffer toelute and remove impurities, after which pertussis toxin (PT) is elutedand isolated with a buffer of appropriate pH and ionic strength. In thecolumn chromatography method, the column is packed with ion exchangegel, through which the starting material, i.e., crudely purifiedpertussis toxin (PT), is passed at a flow rate of 100-500 ml/cm²/hr tocause toxin adsorption. In the batch method, the crudely purifiedpertussis toxin (PT) is placed in a container, to which ion exchange gelis added directly, followed by stirring for about 30 minutes to 3 hours,preferably about 1 hour, to cause toxin adsorption. Such adsorption ofthe crudely purified pertussis toxin (PT) is achieved using a buffer ofa pH level of 5.0-6.0 and an electroconductivity of 100-300 umho(0.1-0.3 mS), e.g., a 0.01-0.02 M phosphate buffer (pH 5.5-6.0). Elutionfrom the ion exchange gel to which the pertussis toxin (PT) has beenadsorbed can be achieved using a buffer of a pH level of 7.0-7.5 and anelectroconductivity of 1,000-2,000 umho (1-2 mS), e.g., a 0.1-0.2 Mphosphate buffer (pH 7.0-7.5). By subjecting the eluate obtained by theabove-described treatment to the aluminum hydroxide gel treatment orzonal centrifugation treatment described below, pertussis toxin (PT)having endotoxin selectively removed can be separated with substantiallyno loss.

In the present invention, the aluminum hydroxide gel treatment forendotoxin removal is carried out to adsorb only the endotoxinselectively by bringing the subject into contact with previouslyprepared aluminum hydroxide gel in the presence of ammonium sulfate.But, the aluminum hydroxide gel, whose amount to be used is less thanone-tenth of that used in WO93/10216, hardly absorbs any amounts ofprotective components of Bordetella pertussis. It is normally preferablethat this treatment be carried out after concentration by a knownmethod, such as ammonium sulfate salting-out or an ultrafiltrationmembrane method. Aluminum ions useful for the previously preparedaluminum hydroxide gel include those of soluble aluminum compounds, suchas aluminum sulfate and aluminum chloride, with preference given to thealuminum ions of aluminum chloride. It is preferable that aluminumhydroxide gel be prepared by adding a 2 M sodium hydroxide solution to a25-190 mM aluminum salt solution (e.g., 0.9-4.5% aluminum chloridesolution) to a pH level of 7.0-7.5, followed by gentle reaction at 4° C.to room temperature for 1 to 3 hours, to form the desired aluminumhydroxide gel. The aluminum hydroxide gel obtained by theabove-described treatment is then treated to recover the resulting gelprecipitate by a known method, such as filtration or centrifugation, toremove free aluminum ions after completion of the reaction. Theprotective component of Bordetella pertussis concentrated by a knownmethod, such as ammonium sulfate salting-out, is recovered bycentrifugation; the precipitate is dissolved in a 0.25 M phosphatebuffer (pH 7.0-7.5) supplemented with 0.25 M sodium chloride. To thisprotective component of Bordetella pertussis, a saturated ammoniumsulfate solution is added to a final concentration of 2.0-8.0 v/v %,followed by addition of previously prepared, recovered aluminumhydroxide gel to a final concentration of 0.1-1.0 mg/ml, preferably0.2-0.5 mg/ml, and gentle reaction at 4° C. to room temperature for 30minutes to 1 hour. After completion of the reaction, the aluminumhydroxide gel is removed by a known method, such as filtration orcentrifugation, to separate the protective component of Bordetellapertussis having endotoxin removed, with substantially no loss.

In the present invention, zonal centrifugation treatment is carried outto remove endotoxin, and is preferably carried out after concentrationby a known method, such as ammonium sulfate salting-out. Zonalcentrifugation methods include sucrose density gradient centrifugation,cesium chloride density gradient centrifugation and potassium tartratedensity gradient centrifugation, with preference given to sucrosedensity gradient centrifugation. For example, when sucrose densitygradient centrifugation is carried out on a sucrose density gradient of0-30 w/v % at an R_(max) of 60,000 to 122,000 G for about 10 to 24hours, the protective component of Bordetella pertussis having endotoxinremoved can be separated.

PT is detoxified by using a conventional detoxification technique asdescribed in British Journal of Experimental Pathslogy, vol. 44, p. 177,(1963). FHA, PRN and FIM may be inactivated, for example, by the methodas described in Japanese Patent Unexamined Publication No. 52726/1989.An improved purified pertussis component vaccine which is superior to aknown pertussis vaccine can be produced by blending in any desirededratio of protective components of Bordetella pertussis obtained by themethod of present invention. Namely, it's not possible to change theratio of each component which is stable in whole cell or co-purifiedacellular vaccine without obtaining furified component respectively,while an antigen ratio can be chosen in the method of present inventionwhich gives the optimal which gives the optimal response in humans as apertussis vaccine since each component is efficiently purified in thepresent invention. The purified pertussis component vaccine is desirableto blend the protective components in as little amount of total proteinas possible and in a way of giving more effective immunogenicity. Thepurified pertussis component vaccine of the present invention preferablyincludes all of three components, i.e. FHA, FIM and PT, and may alsoinclude other pharmaceutically acceptable components such as PRN whichdoes not give undesired side effects.

When blending these components to produce a purified pertussis componentvaccine of the present invention, the ratio of it may be examplified inExamples mentioned hereinafter. The component vaccine of the presentinvention has a PT:FHA:FIM ratio of approximate 4-6:8-10:1, preferably5-6:8-10:1, and comprise, for example, 20-30 μg-protein/ml of PT, 40-50μg-protein/ml of FHA and 5-10 μg-protein/ml of FIM, preferably 25-30μg-protein/ml of PT, 40-50 μg-protein/ml of FHA and 5 μg-protein/ml ofFIM. The component vaccine mentioned above may further include 5-10μg-protein/ml of PRN, and has a PT:FHA:PRN:PT ratio of 2-6:4-10:1-2:1,preferably 5-6:8-10:2:1. Namely, it preferably comprise 25-30μg-protein/ml of PT, 40-50 μg-protein/ml of FHA, 10 μg-protein/ml of PRNand 5 μg-protein/ml of FIM.

The above-described effect of the present invention can be summarized asfollows: The method of the present invention is characterized by the useof the same means of purification for all subject protective componentsof Bordetella pertussis. This obviates the necessity of differentpainstaking procedures for the respective components as in prior artmethods, thus permitting component purification with high efficiency andhigh recovery rate, an aspect very advantageous for industrialproduction. In addition, the endotoxin content, as determined by theLimulus test, is not more than 1 ng per 100 μg total protein, for allprotective components of Bordetella pertussis obtained by the presentinvention, providing very high practical value. It is also possible toproduce an improved purified pertussis component vaccine comprising aneffective combination of pertussis filamentous hemagglutinin (FHA),pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin(PT).

EXAMPLES

The present invention is hereinafter described in more detail by meansof, but is not limited to, the following working examples and referenceexamples. In the following description, pertussis toxin (PT), pertussisfilamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussisfimbriae (FIM) and endotoxin are also referred to as PT, FHA, 69K-OMP,FIM and ET, respectively.

Example 1

Bordetella pertussis Tohama phase I strain was cultured to a finalconcentration of 2 billion cells/ml by Roux bottle stationary culture(450 ml, 35° C., 5 days) and tank agitating culture (40 l, 35° C., 2days) using Stainer-Scholte medium, to yield a Bordetella pertussisculture.

The cell culture was concentrated to a one-tenth volume using anultrafiltration membrane, after which it was centrifuged to separate thesupernatant and cells. To the supernatant, a 1 M phosphate buffer (pH8.0) was added to a final concentration of 0.1 M, followed by additionof an calcium acetate solution to a final concentration of 1.6 w/v % andstirring at room temperature for 1 hour. This calcium phosphate gelsolution was filtered. The resulting filtrate was concentrated anddesalinized to an electroconductivity of 200 umho using anultrafiltration membrane, passed through a sulfopropyl cation exchangechromatography column (produced by Tosoh Corporation), washed with a0.01 M phosphate buffer (pH 6.0), and eluted with a 0.1 M phosphatebuffer (pH 7.0), to yield pertussis toxin (PT). Next, cells weredispersed in a one-tenth volume (relative to the amount of culturebroth) of a 0.05 M phosphate buffer (pH 8.0) supplemented with 1 Msodium chloride, followed by centrifugation to yield the supernatant andcells. To the supernatant, a calcium acetate solution was added to afinal concentration of 0.5 w/v %, followed by stirring at roomtemperature for 1 hour. This calcium phosphate gel solution wasfiltered; the resulting gel layer was collected. The gel layer waseluted with a 0.1 M phosphate buffer (pH 8.0) supplemented with 1 Msodium chloride to yield a solution containing pertussis filamentoushemagglutinin (FHA). Separately, cells were dispersed in a one-tenthvolume (relative to the amount of culture broth) of a 0.01 M phosphatebuffer (pH 7.0) supplemented with 0.15 M sodium chloride, after which itwas heated in 60° C. warm water for 90 minutes, followed bycentrifugation to yield the supernatant. To the supernatant, a 1 Mphosphate buffer (pH 8.0) was added to a final concentration of 0.1 M,after which a calcium acetate solution was added to a finalconcentration of 1.6 w/v %, followed by stirring at room temperature for1 hour. This calcium phosphate gel solution was filtered; the filtrateand the gel layer were collected. The filtrate was concentrated anddesalinized to an electroconductivity of 200 umho using anultrafiltration membrane and passed through a sulfopropyl cationexchange chromatography column (produced by Tosoh Corporation); theeffluent was collected to yield a solution containing pertactin (PRN,69K-OMP). Separately, the gel layer was eluted with a 0.1 M phosphatebuffer (pH 8.0) supplemented with 1 M sodium chloride to yield asolution containing pertussis fimbriae (FIM).

Control sample was prepared as follows: Ammonium sulfate was added at220 g per liter of culture broth, followed by sufficient stirring. Afterbeing kept standing at 4° C. for about 14 days, the mixture wascentrifuged; the supernatant was discarded, and the precipitate wascollected. To the precipitate thus obtained, a one-tenth volume(relative to the amount of culture broth) of a 0.05 M phosphate buffer(pH 8.0) supplemented with 1 M sodium chloride was added, followed bysufficient stirring. After being kept standing at 4° C. for 4 days, themixture was again centrifuged; the supernatant was collected to yield asolution containing pertussis toxin (PT), pertussis filamentoushemagglutinin (FHA), pertactin (PRN, 69K-OMP) or pertussis fimbriae(FIM).

The pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA),pertactin (PRN, 69K-OMP) or pertussis fimbriae (FIM) content in eachsample was determined by ELISA, with purified products of pertussistoxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN,69K-OMP) and pertussis fimbriae (FIM) as references. Results areexpressed in μg protein/ml unit.

Protein content determination: Protein precipitated with heatedtrichloroacetic acid was quantitated by the Lowry method, with bovineserum albumin (Fraction V, produced by Wako Pure Chemical Industries) asa reference. Results are expressed in μg protein/ml unit.

The results for Roux bottle culture broth and those for tank culturebroth are shown in Tables 1 and 2, respectively.

TABLE 1 Active Total Purity (%) Ingredient Protein (active ingredientProtein Content protein Content Recovery* (μg protein/ content/totalSample (μg protein/ml) (%) ml) protein content) PT 2656.8 90.0 2662.199.8 FHA 9161.7 85.0 9339.1 98.1 FIM 474.7 244.6 495.5 95.8 69K-OMP3683.8 244.6 3607.2 102.1 *Each figure represents a percent valuerelative to the control group.

TABLE 2 Active Total Purity (%) Ingredient Protein (active ingredientProtein Content protein Content Recovery* (μg protein/ content/totalSample (μg protein/ml) (%) ml) protein content) PT 3242.2 78.9 3359.896.5 FHA 9527.0 98.0 9752.9 97.7 FIM 675.0 1184.0 714.7 95.0 69K-OMP4333.5 2364.0 4505.1 96.3 *Each figure represents a percent valuerelative to the control group.

It is evident from these figures that each protective component wasefficiently isolated, and that in the case of tank culture broths,pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM), both produced atlow productivity in the case of Roux bottle culture broths, wererecovered in large amounts.

Reference Example 1

To a control solution prepared by the method described in Example 1,calcium acetate was added to a final concentration of 0.5 w/v %,followed by stirring at room temperature for 1 hour. To the filtrateobtained by filtering this calcium solution, a half amount of asaturated ammonium sulfate solution was added; the mixture was keptstanding at 4° C. for 7 days. This ammonium sulfate salting-out productwas centrifuged; the resulting precipitate was collected and resuspendedin a 0.025 M phosphate buffer (pH 7.0) supplemented with 0.25 M sodiumchloride to yield a starting material. To this starting material,aluminum hydroxide gel, previously prepared to a final concentration of0.4 mg/ml, was added; to the aluminum hydroxide gel recovered bycentrifugation, ammonium sulfate was added to a final concentration of0, 2, 4 or 8 w/v %, followed by gentle stirring at room temperature for30 minutes. After completion of the reaction, the aluminum hydroxide gelwas removed by centrifugation to separate the supernatant. Eachsupernatant was assayed for hemagglutination activity and endotoxincontent by the methods described below. The results are shown in Table3.

Determination of hemagglutination activity: After the sample wasserially diluted 2 folds with a 0.01 M phosphate buffered saline, 0.6v/v % chick immobilized red blood cells were added to causehemagglutination. The maximum dilution rate of each sample showinghemagglutination was taken as the hemagglutinin titer HA. Determinationof endotoxin (ET) content: Using Escherichia coli (Difico 055-B5) as areference strain, ET content was determined by the Limulus test (WakoPure Chemical kit). Results are expressed in ng/ml unit. It is evidentfrom Table 3 that endotoxin can be selectively removed, without activeingredient loss, by treating the sample with previously preparedaluminum hydroxide gel in the presence of ammonium sulfate.

TABLE 3 Amount of HA Ammonium Sulfate Endotoxin Recovery Added ContentHA Value Rate* (w/v %) (ng/ml) (HAU/ml) (%) 0 15.8 16000 50.0 2 11.132000 100.0 4 <9.0 32000 100.0 8 <9.0 24000 75.0 *Each figure forendotoxin removal rate or HA recovery rate is a percent value relativeto the pretreatment value.

Example 2

To each of pertussis toxin (PT), pertussis filamentous hemagglutinin(FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM) as obtainedin Example 1, a half amount of a saturated ammonium sulfate solution wasadded, followed by sufficient stirring. After being kept standing at 4°C. for 1 week, the mixture was again centrifuged; the resultingprecipitate was collected.

This precipitate was dissolved in a 0.025 M phosphate buffer (pH 7.0)supplemented with 0.25 M sodium chloride to yield a solution ofpertussis toxin (PT), pertussis filamentous hemagglutinin (FHA),pertactin (PRN, 69K-OMP) or pertussis fimbriae (FIM). To each solution,a saturated ammonium sulfate solution was added to a final concentrationof 4.0 v/v %. To this mixture, previously prepared, recovered aluminumhydroxide gel was added to a final concentration of 0.4 mg/ml, followedby gentle stirring for 30 minutes at room temperature. After completionof the reaction, the aluminum hydroxide gel was removed bycentrifugation to yield pertussis toxin (PT), pertussis filamentoushemagglutinin (FEA), pertactin (PRN, 69K-OMP) and pertussis fimbriae(FIM).

Pertussis toxin (PT) content, pertussis filamentous hemagglutinin (FHA)content, pertactin (PRN, 69K-OMP) content and pertussis fimbriae (FIM)content were determined in the same manner as in Example 1; andendotoxin content, in the same manner as in Reference Example 1. Theresults are shown in Table 4.

TABLE 4 Endotoxin Content Active Ingredient (ng/100 μg Protein ContentRecovery Sample protein) (μg protein/ml) Rate* (%) PT 0.01 2999.0 82.5FHA 0.11 9060.2 95.1 FIM 0.54 478.6 70.9 69K-OMP 0.08 3505.8 80.9 *Eachfigure for recovery rate represents a percent ratio relative to thepretreatment value.

It is evident from this table that endotoxin was selectively removed,with substantially no loss of any protective component, the endotoxincontent per 100 μg protein/ml being not more than 1 ng/ml for allcomponents.

Example 3

To each of pertussis toxin (PT), pertussis filamentous hemagglutinin(FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM) as obtainedin Example 1, a half amount of a saturated ammonium sulfate solution wasadded, followed by sufficient stirring. After being kept standing at 4°C. for 1 week, the mixture was again centrifuged; the resultingprecipitate was collected. This precipitate was dissolved in a 0.05 Mphosphate buffer (pH 8.0) supplemented with 1 M sodium chloride, afterwhich it was dialyzed by the tube method using a 0.05 M phosphate buffer(pH 8.0) supplemented with 1 M sodium chloride as the external fluid, toyield a solution of pertussis toxin (PT), pertussis filamentoushemagglutinin (FHA), pertactin (PRN, 69K-OMP) or pertussis fimbriae(FIM). The concentrate dialyzate was subjected to sucrose gradientdensity centrifugation on a sucrose density gradient of 1-30 w/w % andat an R_(max) of 64,900 G for about 18 hours. After completion of thecentrifugation, 34 w/w % sucrose was fed into the rotor at a low rate ofrotation to collect fractions.

Pertussis toxin (PT) content, pertussis filamentous hemagglutinin (FHA)content, pertactin (PRN, 69K-OMP) content and pertussis fimbriae (FIM)content were determined in the same manner as in Example 1; andendotoxin content, in the same manner as in Reference Example 1. Theresults are shown in Table 5.

TABLE 5 Endotoxin Content Active Ingredient (ng/100 μg Protein ContentRecovery Sample protein) (μg protein/ml) Rate* (%) PT 0.04 231.2 82.5FHA 0.01 849.4 79.3 FIM 0.20 49.1 80.3 69K-OMP 0.03 319.8 92.0 *Eachfigure for recovery rate represents a percent ratio relative to thepretreatment value.

It is evident from this table that endotoxin was selectively removed,with substantially no loss of any protective component, the endotoxincontent per 100 μg protein/ml being not more than 1 ng/ml for allcomponents.

Example 4

To the PT as obtained in Example 3, with addition of amino acid such asLysine, was added formalin to a final concentration of 0.4 v/v %, andafter through mixing, was allowed to stand in an incubator at 39° C. for21-35 days.

To each of FHA, 69K-OMP and FIM as obtained in Example 3, was addedformaline to a final concentration of 0.4 v/v %, and after throughmixing, was allowed to stand in an incubator at 39° C. for 7 days.

Each of these components as treated above was dialyzed against 4 mMphosphate buffer (pH 7.0) supplemented with 0.15M sodium chloride toyield detoxificated PT, inactivated FHA, inactivated 69K-OMP andinactivated FIM.

These detoxificated or inactivated components were blended in severalratios shown in Table 6 and 7, and followed by addition of aluminumchloride to a final concentration of 0.2 mg/ml to give a vaccinerespectively.

The results for the experiments of mouse intracerebral potency withthese blended vaccines, are shown in Table 6 and 7. The experiments wereperformed according to the method of Japanese Minimum Requirements forBiological Products (Association of Biologicals Manufactures of Japan).

TABLE 6 Protein content Mouse intracerebral of respective potencyprotective components 50% effective (μg protein/ml) dose PT FHA FIM69K-OMP IU/ml (μg protein) 10 40 0 0 10.9 1.06 20 30 0 0 13.4 0.89 20 400 0 11.6 1.18 20 50 0 0 13.6 1.18 20 80 0 0 12.6 1.81 30 40 0 0 19.30.85 40 40 0 0 18.7 1.04

TABLE 7 Potein content Mouse intravcerebral of respective potencyprotective components 50% effective (μg protein/ml) dose PT FHA FIM69K-OMP IU/ml (μg protein) 25 25 0 0 19.5 1.18 25 25 5 0 26.0 0.98 25 250 10 24.1 1.18 25 25 5 10 19.3 1.60 25 50 0 0 24.1 1.47 25 50 5 0 22.21.70 25 50 0 10 23.7 1.68 25 50 5 10 24.8 1.71

It is evident from these figures that both inactivated 69K-OMP andinactivated PIM had small effects on the mouse intracerebral potency,and no significant difference were observed among the blended vaccineswhich contain more than 25 μg protein/ml of detoxificated PT.

Example 5

The experiment of mouse aerozol infection protecting potency wereperformed with the blended vaccines as obtained in Example 4. Eachvaccine diluted to one-third was subcutaneously administered to 4week-old mouse respectively with 0.2 ml of each diluted one. Four weekslater after the administration, each mouse was subjected to airwayinfection with 18-323 phase I strain of Bordetella pertussis by usingthe aerozol chamber, and 10 days later after the infection, the abdomenof each mouse was opened and the trachea and lung were picked out fromeach infected mice.

The specimen of each homoginized tissue applied to Bordet-Gengou agar.The agar was cultured at 35° C. for 5 days and the colonies ofBordetella pertussis were counted.

Based on the colony counts of the non-administered mice, the protectivedose was calculated.

The 75% protective dose was calculated in the case of trachea, and the50% protective dose was calculated in the case of lung. Results wereexpressed in μg protein.

And growth inhibitory rate was calculated in the high dose administeredgroup. Equation of the growth inhibitory rate was as follows.

${{Growth}\mspace{14mu} {inhibitory}\mspace{14mu} {rate}\mspace{14mu} (\%)} = {\left( {1 - \frac{{Colony}\mspace{14mu} {counts}\mspace{14mu} {of}\mspace{14mu} {High}\text{-}{dose}\mspace{14mu} {administered}\mspace{14mu} {mice}}{{Colony}\mspace{14mu} {counts}\mspace{14mu} {of}\mspace{14mu} {non}\text{-}{administered}\mspace{14mu} {mice}}} \right) \times 100}$

Results are shown in Table 8.

TABLE 8 Protein content of respective protective Trachea Lungscomponents 75% Protective Growth 50% Protective Growth (μg protein/ml)dose inhibitory dose inhibitory PT FHA FIM 69K-OMP (μg protein) rate (%)(μg protein) rate (%) 40 40 0 0 3.75 97.5 1.00 89.9 20 80 0 0 4.76 87.71.10 92.6 25 50 0 0 4.14 89.5 0.96 89.7 25 50 5 0 1.07 100 0.49 100 2550 0 10 1.74 100 0.49 100 25 50 5 10 0.52 100 0.26 100

It is evident from this table that both inactivated 69K-OMP andinactivated FIM ahad small effects on the mouse intracerebral potency,but they showed protective effect on the aerozol infection potency.

Test Example 1

Differences of adsorption performance between the calcium phosphate gel(In-side Gel) forming and the hydroxyapatite gel (Out-side Gel) on FHAand FIM.

Roux bottle stationary culture prepared by method described inExample 1. The cell culture was concentrated to a one-tenth volume usingan ultrafiltration membrane, after which it was centrifuged to separatethe supernatant (Sample a) and cells. The cells were dispersed in aone-tenth volume of 0.05 M phosphate buffer (pH 8.0) supplemented with 1M sodium chloride and stirred well. It was kept standing at 4° C. for 4days, followed by centrifugation to yield the eluted supernatant (Sampleb) included PT, FHA, 69K-OMP and FIM.

The culture supernatant (Sample a) and eluted supernatant (Sample b)described above were treated as following 1) or 2).

1) Calcium Phosphate Gel (In-Side Gel) Forming Treatment

To the samples, a 1 M phosphate buffer (pH 8) was added, followed byaddition of a calcium acetate solution to a final concentration of 0.5w/v %, 1.0 w/v % or 2.0 w/v % and gently stirred at room temperature for1 hour, followed by centrifugation at 1000 rpm for 10 minutes tosupernatant and gel residue. The eluted solution was obtained by elutingthe gel residue with a 0.1 M phosphate buffer (pH 8.0) supplemented witha 1M sodium chloride.

2) Hydroxyapatite Gel (Out-Side Gel) Treatment

Hydroxyapatite gel (produced by BDH Chemicals Ltd) was equilibrated witha 0.01M phosphate buffer (pH 8.0). The gel was added to 20 w/v %, 10 w/v% or 50 w/v % to the sample volume and gently stirred at roomtemperature for 1 hour, followed by centrifugation at 1000 rpm for 10minutes to separate supernatant from gel residue. The eluted solutionwas obtained by eluting the gel residue with a 0.1 M phosphate buffer(pH 8.0) supplemented with a 1M sodium chloride.

The content of FHA or FIM content in each sample was determined byELISA, with FHA or FIM as the house references. The assay results areexpressed in μg protein/ml unit. Protein content; Protein precipitatedwith heated trichloroacetic acid was quantitated by the Lowry method,with bovine serum albumin (Fraction V, produced by Wako Pure ChemicalIndustries) as a reference. Results are expressed in μg protein/ml unit.

Adsorption rate and recovery rate to the gel on FHA and FIM werecalculated by following equations respectively.

${{Adsorption}\mspace{14mu} {rate}\mspace{14mu} (\%)} = {\left( {1 - \frac{{Supernatant}\mspace{14mu} {of}\mspace{14mu} {post}\text{-}{gel}\mspace{14mu} {treatment}}{{Pre}\text{-}{gel}\mspace{14mu} {treatment}\mspace{14mu} {Sample}}} \right) \times 100}$${{Recovery}\mspace{14mu} {rate}\mspace{14mu} (\%)} = {\frac{{Eluted}\mspace{14mu} {solution}\mspace{14mu} {of}\mspace{14mu} {post}\text{-}{gel}\mspace{14mu} {treatment}}{{Pre}\text{-}{gel}\mspace{14mu} {treatment}\mspace{14mu} {Sample}} \times 100}$

Results are shown in Table 9 and Table 10

TABLE 9 a) Culture supernatant FIN FHA Re- Absorption RecoveryAbsorption covery rate (%) rate (%) rate (%) rate (%) Concentration 0.589.7 69.6 0.0 0.0 of calcium 1.0 90.4 63.0 95.0 77.9 acetate added 2.089.5 44.4 94.3 98.4 (w/v %) Concentration 2.0 25.4 27.5 0.0 0.0 of 10.049.5 26.4 7.9 4.1 hydroxyapatite 50.0 90.8 48.9 22.1 5.3 added (w/v %)

TABLE 10 b) The eluted solution from the cell with 0.05M phosphatebuffer (pH 8.0) supplemented with 1M-NaCl FIN FHA Re- AbsorptionRecovery Absorption covery rate (%) rate (%) rate (%) rate (%)Concentration 0.5 96.3 87.2 18.7 12.5 of calcium 1.0 98.7 61.0 99.9 74.3acetate added 2.0 98.7 55.1 99.8 94.3 (w/v %) Concentration 2.0 10.8 1.04.6 1.1 of 10.0 4.7 3.8 9.7 3.9 hydroxyapatite 50.0 15.6 16.7 13.5 8.2added (w/v %)

The calcium phosphate gel (In-side gel) strongly adsorbs both FHA andFIM, but the hydroxyapatite gel has small adsorption effect on the FIM.Also the Hydroxyapatite gel compared with the calcium phosphate gel, hasless adsorption effect on the FHA and depend on the volume added.

INDUSTRIAL APPLICABILITY

The method of the present invention is characterized by the use of thesame means of purification for all subject protective components ofBordetella pertussis. Each component can therefore be purified with highefficiency and high recovery rate, an aspect very advantageous forindustrial production. It is also possible to efficiently produce animproved purified pertussis component vaccine comprising an effectivecombination of pertussis filamentous hemagglutinin (FHA), pertactin(PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT).

1-15. (canceled)
 16. A pertussis vaccine comprising the protectivecomponents of Bordetella pertussis pertussis toxin (PT) and pertussisfilamentous hemagglutinin (FHA), wherein said components are admixed ina weight ratio of 1:1 μg protein, and said vaccine further comprises aprotective component pertactin (PRN) of Bordetella pertussis.
 17. Thepertussis vaccine according to claim 16, wherein said components areadmixed in a weight ratio of about 1:1:0.4 μg protein of PT:FHA:PRN. 18.The pertussis vaccine according to claim 16, further comprising theprotective component pertussis fimbiaie (FIM) of Bordetella pertussis.19. The pertussis vaccine according to claim 18, wherein said componentsare admixed in a weight ratio of about 1:1:0.2-0.4:0.2-0.4 μg protein ofPT:FHA:PRN:FIM.
 20. The pertussis vaccine according to claim 19, whereinsaid components are admixed in a weight ratio of about 1:1:0.4:0.2 μgprotein of PT:FHA:PRN:FIM.
 21. A pertussis vaccine comprising theprotective components of Bordetella pertussis PT and FHA, wherein saidcomponents are admixed in a weight ratio of 1:1 μg protein, and saidvaccine further comprises a protective component FIM of Bordetellapertussis.
 22. The pertussis vaccine according to claim 21, wherein saidcomponents are admixed in a weight ratio of about 1:1:0.2 μg protein ofPT:FHA:FIM.
 23. The pertussis vaccine according to claim 16, whereinsaid components are admixed in a weight ratio of 1:1:0.2-0.4 μg proteinof PT:FHA:PRN.
 24. The pertussis vaccine according to claim 23, whereinsaid components are admixed in a weight ratio of 1:1:0.4 μg protein ofPT:FHA:PRN.
 25. The pertussis vaccine according to claim 18, whereinsaid components are admixed in a weight ratio of 1:1:0.2-0.4:0.2-0.4 μgprotein of PT:FHA:PRN:FIM.
 26. The pertussis vaccine according to claim25, wherein said components are admixed in a weight ratio of 1:1:0.4:0.2μg protein of PT:FHA:PRN:FIM.
 27. The pertussis vaccine according toclaim 21, wherein said components are admixed in a weight ratio of1:1:0.2-0.4 μg protein of PT:FHA:FIM.
 28. The pertussis vaccineaccording to claim 27, wherein said components are admixed in a weightratio of 1:1:0.2 μg protein of PT:FHA:FIM.